Bioseparation Technology Laboratory, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Drive, Building 10, Room 8N230, Bethesda, MD 20892-1762, USA.
J Chromatogr A. 2011 Sep 9;1218(36):6128-34. doi: 10.1016/j.chroma.2010.11.014. Epub 2010 Nov 21.
The performance of the figure-8 column configuration in centrifugal counter-current chromatography was investigated by changing the angle between the column axis (a line through the central post and the peripheral post on which the figure-8 coil is wound) and the centrifugal force. The first series of experiments was performed using a polar two-phase solvent system composed of 1-butanol-acetic acid-water (4:1:5, v/v) to separate two dipeptide samples, Trp-Tyr and Val-Tyr, at a flow rate of 0.05 ml/min at 1000 rpm. When the column angle was changed from 0° (column axis parallel to the centrifugal force) to 45° and 45° to 90° (column axis perpendicular to the centrifugal force), peak resolution (Rs) changed from 1.93 (Sf=37.8%) to 1.54 (Sf=30.6%), then to 1.31 (Sf=40.5%) with the lower mobile phase and from 1.21 (Sf=38.8%) to 1.10 (Sf=34.4%), then to 0.99 (Sf=42.2%) with the upper mobile phase, respectively, where the stationary phase retention, Sf, is given in parentheses. The second series of experiments was similarly performed with a more hydrophobic two-phase solvent system composed of hexane-ethyl acetate-methanol-0.1M hydrochloric acid (1:1:1:1, v/v) to separate three DNP-amino acids, DNP-glu, DNP-β-ala and DNP-ala, at a flow rate of 0.05 ml/min at 1000 rpm. When the column angle was altered from 0° to 45° and 45° to 90°, Rs changed from 1.77 (1st peak/2nd peak) and 1.52 (2nd peak/3rd peak) (Sf=27.3%) to 1.24 and 1.02 (Sf=35.4%), then to 1.69 and 1.49 (Sf=42.1%) with the lower mobile phase, and from 1.73 and 0.84 (SF=41.2%) to 1.44 and 0.73 (Sf=45.6%), then to 1.21 and 0.63 (Sf=55.6%) with the upper mobile phase, respectively. The performance of figure-8 column at 0° and 90° was also compared at different flow rates. The results show that Rs was increased with decreased flow rate yielding the highest value at the 0° column angle with lower mobile phase. The overall results of our studies indicated that a 0° column angle for the figure-8 column enhances the mixing of two phases in the column to improve peak resolution while decreasing the stationary phase retention by interrupting the laminar flow of the mobile phase.
研究了通过改变柱轴（通过中心柱和外周柱的线，其上缠绕着 8 字形线圈）与离心力之间的角度来改变离心逆流色谱中 8 字形柱的配置性能。第一系列实验是在极性两相溶剂系统中进行的，该系统由 1-丁醇-乙酸-水（4：1：5，v/v）组成，以 0.05 ml/min 的流速在 1000 rpm 下分离两种二肽样品，色氨酸-酪氨酸和缬氨酸-酪氨酸。当柱角从 0°（柱轴与离心力平行）变为 45°和 45°变为 90°（柱轴与离心力垂直）时，峰分辨率（Rs）从 1.93（ Sf = 37.8%）变为 1.54（ Sf = 30.6%），然后变为 1.31（ Sf = 40.5%），而较低的流动相从 1.21（ Sf = 38.8%）变为 1.10（ Sf = 34.4%），然后变为 0.99（ Sf = 42.2%），其中固定相保留率 Sf 在括号中给出。第二系列实验同样使用更疏水的两相溶剂系统进行，该系统由正己烷-乙酸乙酯-甲醇-0.1M 盐酸（1：1：1：1，v/v）组成，以 0.05 ml/min 的流速在 1000 rpm 下分离三种 DNP-氨基酸，DNP-谷氨酸，DNP-β-丙氨酸和 DNP-丙氨酸。当柱角从 0°变为 45°和 45°变为 90°时，Rs 从 1.77（第一个峰/第二个峰）和 1.52（第二个峰/第三个峰）（ Sf = 27.3%）变为 1.24 和 1.02（ Sf = 35.4%），然后变为 1.69 和 1.49（ Sf = 42.1%），较低的流动相，从 1.73 和 0.84（SF = 41.2%）变为 1.44 和 0.73（SF = 45.6%），然后变为 1.21 和 0.63（SF = 55.6%），较高的流动相。还比较了 0°和 90°处 8 字形柱在不同流速下的性能。结果表明，随着流速的降低，Rs 增加，在较低流动相的 0°柱角处获得最高值。我们研究的总体结果表明，8 字形柱的 0°柱角增强了柱中两相的混合，从而提高了峰分辨率，同时通过中断流动相的层流降低了固定相保留率。
